1. Field of the Invention
The present invention generally relates to the crystallization of macromolecules such as proteins from solutions. The present invention is particularly related to the preparation of arrays of solutions useful for screening crystallization conditions to determine which conditions are optimal for the crystallization of macromolecules. Even more particularly, the present invention is related to preferred techniques of preparing solutions suitable for screening, and for the screening conditions themselves, where the total macromolecule solution volume is very small, typically in nanoliter quantities or below.
2. Background of the Invention
The crystallization of macromolecules, especially of biological macromolecules, is an important activity in many fields. Obtaining high quality crystals of any given macromolecule typically enables subsequent solution of the macromolecule's three dimensional structure (atomic configuration) using diffraction techniques. Of particular interest, the three dimensional structures so obtained can be of paramount importance in the rational design of drugs or other therapeutics. Additionally, it is commonly accepted that one of the primary benefits that will flow from elucidation of the genome will be an improved understanding gained of the proteome, the entire set of expressed proteins in a particular biological organism. However, the full advantage that can be gained from that improved understanding of the proteome can only be realized with the knowledge of the three dimensional atomic configuration of each substituent protein. For example, a knowledge of the three dimensional atomic configuration of a given protein, referred to herein as its active conformation, will provide opportunities to use computerized methods and techniques, among others, to design drug molecules that will effectively and/or efficiently interact with the target protein.
The orderly crystallization of macromolecules from a solution containing the macromolecules results from complex interplay between many variables, including process kinetics (the rate at which the solution approaches supersaturation conditions), pH, ionic composition of buffer components in the solution, the type and concentration of crystallization agents, temperature, etc. As a result, the process of determining what conditions are suitable for crystallizing a given macromolecule is formidable. Typically, hundreds or thousands of experiments are conducted in which one or more of these variables is different from those of one or more of the others. This process, often referred to as screening, is useful to identify the best conditions to use in order to obtain high quality crystals. Usually, once screening has identified suitable conditions, further experiments are performed with those conditions so identified to obtain large crystals for subsequent diffraction studies.
The advent of the genomics and proteomics age has resulted in an unprecedented increase in the number of biological macromolecules available for crystallization. However, due to technical difficulties in expressing and purifying many proteins, the amount of protein available for screening crystallization conditions is often not adequate to provide for screening a suitable number of crystallization conditions if conventional methods of screening are used. This can be illustrated by typical circumstances encountered where only sub-milligram amounts of proteins are available, but it is desirable to screen hundreds or thousands of different conditions. For screening such a large number of conditions with such a small amount of a protein, each screening experiment must use no more than a nanogram of protein. Ideally, each screening experiment would use even less protein. As typical supersaturation levels of proteins in solution are much higher than those corresponding to small molecular weight compounds, the volume of the solution in which the experiment must be carried out, in order to achieve supersaturating concentrations, must be very small. Examples of the small volumes required are typically less than or equal to a few hundred, a hundred, fifty, twenty-five, ten, five or one nanoliter(s).
Conducting screening experiments of macromolecules, in particular of those biological macromolecues, are further constrained by factors in addition to the quantity of protein required. These further constraints include those related to the types of solutions used, the accuracy requirements, the means necessary to dispense small volumes of various components, and limitations on the techniques that can be used for practical manipulation of small volumes. Each of these additional constraints must be overcome in order to allow the screening in low volumes to lessen the quantity of protein required. It would be further desirable to provide such methods that address each of these particular constraints in a manner that allows high levels of flexibility, accuracy, precision and reproducibility. Furthermore, it would be an advantage if the methods so provided were simple to use.